Molecular Genetics of Hemoglobinopathies

Question 1

Hemoglobin Structure - Function Relationships

Answer 1

The major form of adult hemoglobin (HbA) is a α2β2 tetramer of two α- and two β-globin chains. Each globin contains one heme group with a covalently linked iron that binds oxygen, so one HbA can simultaneously bind four oxygen molecules.

Question 2

All of α and α-like genes are in the α-cluster on chromosome _______ while all of β and β-like genes are in the β-cluster on chromosome _______

Answer 2

16

11

Question 3

There are _______ copies of α in the α-cluster but only _______ of β in the β-cluster

Answer 3

two

only one

Question 4

The genes in each cluster have the same _______. At the molecular level the _______ of these genes are very similar

Answer 4

• 5’-to-3’ transcriptional orientation

- promoter and enhancer regions

Question 5

Pseudogene:

Answer 5

resembles a gene but makes no protein. ψζ, ψα, and ψβ are all pseudogenes.

Question 6

The 5’-to-3’ spatial order of genes within each cluster coincides with _______. The sequential expression of these genes during development is under the regulation of the _______, which is located at the most _______ region of each cluster.

Answer 6

• the temporal order of their expression during development
• Locus Control Region (LCR)
• upstream

Question 7

It is currently thought that the _______ and a particular globin gene affects its expression

Answer 7

distance between the LCR and a particular globin gene

Question 8

The LCR presumably makes physical contact with the _______ via _______ to influence gene expression

Answer 8

• promoter and/or negative regulatory regions

- specific transcriptional factors

Question 9

Deletions of the entire LCR of the beta cluster cause _______, a condition in which _______

Answer 9

beta-thalassemias

zero β-globin synthesis leads to precipitation of the α-globin chains.

Question 10

Adults have two Hb forms (made in the bone marrow):

Answer 10

• major form: HbA = α2β2 (97%)
• minor form : HbA2 = α2δ2 (2%)
• Note that δ level is much lower than β because δ has a weaker promoter.

Question 11

Embryonic hemoglobins (made in the yolk sac):

Answer 11

ζ2ε2 = Hb Gower I 
α2ε2 = Hb Gower II 
ζ2γ2 = Hb Portland

Question 12

Fetal hemoglobins (made in the liver):

Answer 12

α2γ2 = HbF

Question 13

Globin Switching

Answer 13

(i) turn-off of ζ and ε, turn-on of α and γ during early embryogenesis.
(ii) turn-off of γ, turn-on of β and δ around the time of birth.

Question 14

Significance of Globin Switching

Answer 14

HbF has higher affinity for O2 at low pO2 than HbA. Thus, HbF in fetal blood is better suited to bind O2 at the placenta (lower pO2) than HbA, which binds O2 at the lung (higher pO2).

Question 15

Structural Variants (qualitative hemoglobinopathies)

Answer 15

Mutations that alter the globin polypeptide properties without affecting its synthesis. Most structural variants are found in the β-globin chain. These mutations may alter O2 binding such as HbKemsey (too tight) and HbKansas (too weak), cause heme loss and denaturation of Hb, or make Hb less soluble such as HbS and HbC. Some can lead to serious red blood cell (RBC) diseases

Question 16

Thalassemias (quantitative hemoglobinopathies)

Answer 16

Disorders of imbalanced globin levels resulted from markedly reduced or no synthesis of one globin type. Can be caused by virtually all types of genetic mutations including deletions, missense and nonsense mutations, and defective transcriptional control

Question 17

Hereditary Persistence of Fetal Hemoglobin (HPFH) `

Answer 17

HPFH is a group of clinically benign conditions that impair the perinatal switch from γ- to β-globin synthesis, leading to continued high-level production of HbF in adults

Question 18

Sickle cell anemia (HbSS)

Answer 18

Most common among people of African origin, where carrier frequency is ~10%. Single base mutation at codon#6 in the β-globin gene changes glutamate to valine. HbS is 80% less soluble than HbA when not bound to O2, and polymerizes into long fibers that distort the RBC into a characteristic sickle shape. These sickled cells become lodged in the micro-capillaries and further exacerbate the sickling crisis.

Question 19

Hemoglobin C disease (HbCC)

Answer 19

A milder form of hemolytic anemia than sickle cell anemia. Caused by a single base mutation at codon#6 of the β-globin gene, changing glutamate to lysine. HbC is less soluble than HbA and tends to form crystals, reducing the deformability of RBC.

Question 20

HbS or HbC trait

Answer 20

Both sickle cell anemia and hemoglobin CC disease are of autosomal recessive inheritance. Sickle cell trait (HbS trait) or hemoglobin C trait (HbC trait) describes the conditions expressed in individuals who are heterozygous of HbS/HbA and HbC/HbA, respectively. They are clinically normal except when under severe low pO2 stress.

Question 21

Hemoglobin SC disease

Answer 21

Compound heterozygotes (βS/βC) have a milder anemia than sickle cell disease.

Question 22

HbS Diagnosis Using RFLP

Answer 22

A recognition site (CCTNAGG) of the restriction enzyme MstII is destroyed in exon 1 by the A-to-T change in the βS mutant allele. The normal allele βA gives 1.15 kb + 0.20 kb fragments, whereas the βS mutant allele give one1.35 kb fragment.

Question 23

A functional Hb tetramer is composed of _______. Homotetramers (e.g. α4, β4, γ4) are _______ O2 carriers and precipitate _______

Answer 23

• two α (or α-like) chains and two β (or β-like) chains
• poor
• inside the RBC

Question 24

Thalassemias are caused by

Answer 24

an imbalance in the relative levels of the α and β globin chains, which leads to the precipitation of the globin in excess and decreases the life span of the RBC

Question 25

α-Thalassemias

Answer 25

Mostly caused by deletions of one or both copies of the α-globin gene in the α-cluster. Thus, γ- and β-globin are in excess. Affects the formation of both fetal and adult hemoglobins.

Question 26

α-thal-1 allele (- -)

Answer 26

Common in Southeast Asia. Caused by deletion of both copies of α-globin genes in the α- cluster. Homozygous state (- -/- -) results in “hydrops fetalis” (stillborn). Most fetal hemoglobin is γ4 (Hb Bart’s) although there is enough ζ2γ2 (Hb Portland) to sustain fetal development. Heterozygotes (αα/–) have mild anemia, a.k.a. α -thalassemia-1 trait.

Question 27

α-thal-2 allele (α -)

Answer 27

Common in Africa, Mediterranean, and Asia. Deletion of one of the two α-globin genes in the α-cluster. 50% decrease in α-globin synthesis. No disease phenotype in heterozygote (αα/α-, silent carrier). Mild anemia in homozygotes (α-/α-), a.k.a. α -thalassemia-2 trait.

Question 28

α-thal-1/α-thal-2 (α -/- -)

Answer 28

Compound heterozygous individuals with only 25% of normal α-globin level. Severe anemia. a.k.a. HbH disease. About 5-30% of their hemoglobin is β4 (HbH), which precipitates.

Question 29

β-Thalassemias

Answer 29

Show a wide range of severity and can be cause by virtually every possible type of mutation in the β-globin gene. High allelic heterogeneity means most patients with β-thalassemia are compound heterozygotes carrying two different mutant alleles of the β-globin gene.

Question 30

Thalassemia major

Answer 30

Characterized by severe anemia, in which most RBCs are destroyed before being released into the circulation. Thinning bone cortex, enlarged liver and spleen resulted from massive effort of blood production at these sites. Treat temporarily with blood transfusions; however, iron accumulation from repeated transfusion leads to organ failure. Iron chelation therapy (e.g. desferrioxamine) is used to reduce the complications of iron overload.

Question 31

Thalassemia minor

Answer 31

Clinically normal, carriers of one β-thalassemia allele

Question 32

Simple β-thalassemia

Answer 32

Caused by mutations or deletions that impair the production of the β-globin chain alone. Other genes in the β-globin cluster unaffected.

Question 33

Complex thalassemia

Answer 33

Caused by large deletions that remove the β-globin gene plus other genes in the β-cluster, or the LCR. Note that some deletions within β-cluster cause HPFH instead of thalassemia

Question 34

β+-thalassemia

Answer 34

Most common form of β-thalassemia (90% of the cases). Some β-globin is made so that some HbA is present. Decrease in β-globin synthesis can be caused by mutations affecting transcription, RNA processing, or protein stability

Question 35

β0-thalassemia

Answer 35

Zero β-globin synthesis so that no HbA is present. Caused by deletion of the β-globin gene, nonsense or frameshift mutations at the 5’ of the coding region that lead to an early stop codon, or mutations that result in no RNA synthesis. [Hb] is ~5% of normal level (of which 95% is α2γ2 with 5% α2δ2), which is not enough for good survival

Question 36

δβ0-thalassemia

Answer 36

No δ or β synthesis due to deletion of the coding sequences for both δ- and β-globin. Milder clinical phenotype than β0-thalassemia because the remaining γ gene(s) is still active after birth instead of switching off as would normally occur. Therefore, HbF (α2γ2) compensates for the absence of HbA (α2β2), about 5-18% of normal level of total hemoglobin production.

Question 37

People with deletions that remove the δ and β genes ( γ is intact) may have either _______, depending on the range of the deletion

Answer 37

δβ 0- thalassemia or HPFH

Question 38

HPFH (hereditary persistent fetal hemoglobin)

Answer 38

No δ or β synthesis because of deletions of both genes.

Question 39

Increased γ-globin expression caused by either of the two following mechanisms:

Answer 39

(1) extended deletion of additional downstream sequences, which likely brings a cis-acting enhancer element closer to the γ-globin gene, or
(2) mutations in the promoter region of one of the two γ-globin genes that destroy the binding site of a repressor, thereby relieving postnatal repression of γ.

Question 40

HPFH individuals are _______, since _______. 100% of hemoglobin is _______. HPFH individuals have _______ HbF level than δβ0-thalassemia individuals

Answer 40

• disease free
• adequate levels of γ chains are still made due to the disruption of the perinatal globin switch from γ to β
• HbF (α2γ2)
• higher